Benchmarking DNA isolation methods for marine metagenomics

Abstract

Metagenomics is a powerful tool to study marine microbial communities. However, obtaining high-quality environmental DNA suitable for downstream sequencing applications is a challenging task. The quality and quantity of isolated DNA heavily depend on the choice of purification procedure and the type of sample. Selection of an appropriate DNA isolation method for a new type of material often entails a lengthy trial and error process. Further, each DNA purification approach introduces biases and thus affects the composition of the studied community. To account for these problems and biases, we systematically investigated efficiency of DNA purification from three types of samples (water, sea sediment, and digestive tract of a model invertebrate Magallana gigas) with eight commercially available DNA isolation kits. For each kit-sample combination we measured the quantity of purified DNA, extent of DNA fragmentation, the presence of PCR-inhibiting contaminants, admixture of eukaryotic DNA, alpha-diversity, and reproducibility of the resulting community composition based on 16S rRNA amplicons sequencing. Additionally, we determined a "kitome", e.g., a set of contaminating taxa inherent for each type of purification kit used. The resulting matrix of evaluated parameters allows one to select the best DNA purification procedure for a given type of sample.

Figure 1

A scheme representing the methodology of the study. Three types of samples were collected. Each sample was processed in three technical replicates with eight commercial DNA purification kits, in addition two types of negative controls (No-input NC; mQ NC) were processed in one replicate. Additional purification steps were applied to some of the resulting DNA samples to follow the removal of contaminants and reduction of eukaryotic DNA load. All samples were evaluated for an indicated set of parameteres to select the best DNA purification strategy.

Figure 2

(A) Total DNA amounts purified by different kits from three types of samples. (B) DNA integrity numbers (DINs) obtained with three types of samples subjected to purification by different kits. Data for three technical replicates are shown. Right—kit ranking (a sum of ranks for different sample types). Lower ranks indicate higher DNA yeild. (5*) DNA extracted from gut flora samples with a Stool kit was of low quality and was heavily contaminated with RNA.

Figure 3

The presence of DNA inhibitors estimated as a dilution factor required to achieve visible production of a 16S rRNA amplicon. Mean results obtained for the three technical replicates are shown.

Figure 4

Eukaryotic DNA admixture estimated by the qPCR Ct values obtained with 18S and 16S rRNA gene-specific primers. Data for three technical qPCR replicates for each of the three kit purification replicates are shown. Rigth—total kit rankings (a sum of ranks for different sample types). Lower ranks indicate higher proportion of microbial DNA.

Figure 5

Determination of kitomes of commercial DNA extraction kits. (A) Diversity of kitomes on a genus level. Data is shown for genera with relative abundances > 5%. (B) Left—contamination levels of natural samples processed with commercial kits. Means of three technical replicates + /- SD are shown. Kit rankings for each sample type are shown above the bar plot. Right—total kit ranking (a sum of ranks for different sample types). Lower ranks indicate lower contamination levels.

Figure 6

Reproducibility of DNA extraction kits and the effect of kits on alpha-diversity of bacterial communities. (A) Reproducibility levels of commercial DNA extraction kits for different sample types. Right—total kit rankings (a sum of ranks for different sample types). Lower rankings indicate higher reproducibility levels. (B) Bar plots representing fractions of OTUs shared between all DNA extraction kits benchmarked for a particular sample type (Universal OTUs, orange), OTUs found by just one kit (Unique OTUs, pink), and other OTUs (found in DNA prepared by two to seven kits, light blue). (C) Fractions of OTUs shared between different numbers of DNA extraction kits. Numbers of OTUs found by all 8 studied DNA extraction kits are shown. (D) Microbial alpha diversity (the Shannon index) of DNA samples prepared using different kits. Data for three technical replicates are shown. Right—total kit rankings (a sum of ranks for different sample types). Lower rankings indicate higher Shannon index values. (E) Contamination level and alpha diversity (Shannon index) of samples processed with different DNA extraction kits. A group of samples with high diversity and low contamination levels is marked with a dashed rectangle.

Figure 7

Effects of DNA extraction kits on the composition of bacterial communities. (A) Microbial communities’ composition of environmental samples at the order level after the decontamination step. Data is shown for all technical replicates independently. Orders with relative abundances > 1% are shown. (B) NMDS plots with points representing microbial communities. Dashed ellipses indicate sample groups distant from the majority of samples.

Figure 8

Radar-plots demonstrating the performance of DNA extraction kits with Sediment (A), Water (B), and M. gigas gut flora samples (C). ν(DNA)—DNA yield, DIN—DNA integrity, Inh—presence of PCR inhibitors (higher rank indicates the lower level of inhibitors), 18S/16S–18S/16S ratio (higher rank indicates the lower ratio), Cont—contamination level (higher rank indicates the lower level of contamination), Rep—reproducibility level, α—alpha-diversity. Kits were ordered by the sum of rankings.